of 10
Ultrastructure of
Shewanella oneidensis
MR-1
nanowires revealed by electron cryotomography
Poorna Subramanian
a,1
, Sahand Pirbadian
b,1
, Mohamed Y. El-Naggar
b,c,d,2
, and Grant J. Jensen
a,e,2
a
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125;
b
Department of Physics and Astronomy, University
of Southern California, Los Angeles, CA 90089;
c
Department of Chemistry, University of Southern California, Los Angeles, CA 90089;
d
Molecular and
Computational Biology Section, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089; and
e
Howard Hughes Medical
Institute, California Institute of Technology, Pasadena, CA 91125
Edited by E. Peter Greenberg, University of Washington, Seattle, WA, and approved February 21, 2018 (received for review November 6, 2017)
Bacterial nanowires have garnered recent interest as a proposed
extracellular electron transfer (EET) pathway that links the bacterial
electron transport chain to solid-phase electron acceptors away
from the cell. Recent studies showed that
Shewanella oneidensis
MR-1 produces outer membrane (OM) and periplasmic extensions
that contain EET components and hinted at their possible role as
bacterial nanowires. However, their fine structure and distribution
of cytochrome electron carriers under native conditions remained
unclear, making it difficult to evaluate the potential electron trans-
port (ET) mechanism along OM extensions. Here, we report high-
resolution images of
S. oneidensis
OM extensions, using electron
cryotomography (ECT). We developed a robust method for fluores-
cence light microscopy imaging of OM extension growth on electron
microscopy grids and used correlative light and electron microscopy
to identify and image the same structures by ECT. Our results reveal
that
S. oneidensis
OM extensions are dynamic chains of intercon-
nected outer membrane vesicles (OMVs) with variable dimensions,
curvature, and extent of tubulation. Junction densities that poten-
tially stabilize OMV chains are seen between neighboring vesicles in
cryotomograms. By comparing wild type and a cytochrome gene
deletion mutant, our ECT results provide the likely positions and
packing of periplasmic and outer membrane proteins consistent
with cytochromes. Based on the observed cytochrome packing den-
sity, we propose a plausible ET path along the OM extensions in-
volving a combination of direct hopping and cytochrome diffusion.
A mean-field calculation, informed by the observed ECT cytochrome
density, supports this proposal by revealing ET rates on par with a
fully packed cytochrome network.
extracellular electron transport
|
electron cryotomography
|
membrane
cytochromes
|
bacterial nanowires
|
Shewanella
R
edox reactions are essential to all biological energy conver-
sion strategies (1). In respiratory organisms, free energy is
harvested from the environment as electrons extracted from an
electron donor are transferred through the cellular electron trans-
port (ET) chain to a terminal electron acceptor (EA). While most
eukaryotes, including humans, are dependent on molecular oxygen
(O
2
) as their terminal EA, anaerobic prokaryotes can acquire en-
ergy by employing a wide variety of alternative EAs. Like O
2
,many
of these EAs can diffuse inside the cell, where they participate in
redox reactions with intracellula
r ET chain components. However,
dissimilatory metal-reducing bacteria (DMRB) can also utilize in-
soluble EAs such as metal oxide minerals that are inaccessible to
the electron transport chain components at the inner membrane, by
transporting electrons across the cell envelope (2
6). This extra-
cellular electron transport (EET) process has important implica-
tions in renewable energy technologies, wastewater treatment,
bioremediation, and global biogeochemical cycles (3, 7
9).
The gram-negative bacteria
Geobacter
and
Shewanella
are two
of the best-studied DMRB model systems (2, 5, 6, 10, 11) and are
known to produce extracellular appendages proposed to act as
bacterial nanowires, transporting electrons over micrometer-long
distances to terminal extracellular EAs.
Geobacter
nanowires are
type IV pili (12) and their electron conductivity has been at-
tributed to either an incoherent electron hopping mechanism
along a path of aromatic residues (13
15) or a coherent
metallic-
like
mechanism facilitated by proposed
π
stacking of aromatic
residues (16
18). These pili may also interact with separate ex-
tracellular redox proteins, possibly working in concert to allow
EET, with the pili playing a larger role at cellular layers more
distant from electrode surfaces (19). Electrochemical gating sig-
natures of transverse conduction through
Geobacter
biofilms that
span interdigitated electrodes appear consistent with a network of
redox cofactors such as the hemes of cytochromes abundant in
DMRB (20), but these measurements do not necessarily preclude
a role for pili in vertical charge transport, especially at biofilm
layers farther away from the underlying electrodes as described
by Steidl et al. (19). Transport through the extracellular ap-
pendages of
Shewanella
requires the presence of multiheme cy-
tochromes as the electron carriers (21), but a detailed analysis of
the underlying mechanism and extent to which it may allow EET
under physiological conditions requires a better understanding of
the cytochrome distribution and structure of the appendages
under native conditions.
Previous electrochemical, biochemical, genetic, and structural
studies of
Shewanella
have identified an intricate network of
redox proteins that traffic electrons from the inner membrane
quinone pool through the periplasm and across the outer
membrane (OM) (6, 7, 11). A critical electron transfer module is
Significance
Recent findings from in vivo fluorescence and immunolabeling
measurements hinted at the possible role of outer membrane
(OM) extensions as
Shewanella oneidensis
MR-1 nanowires.
However, a detailed understanding of the architecture and
electron transport mechanism along OM extensions was lack-
ing. In this work, we report a unique setup for correlative light
and electron microscopy of
Shewanella
OM extensions and
demonstrate that they are chains of interconnected outer
membrane vesicles with densities, consistent with periplasmic
and OM cytochromes, distributed along their length. We pro-
pose, based on the packing density of cytochromes measured
from electron cryotomograms, that the electron transport
mechanism involves a combination of direct electron hopping
and diffusion of electron carriers.
Author contributions: P.S., S.P., M.Y.E.-N., and G.J.J. designed research; P.S. and S.P. per-
formed research; P.S. and S.P. analyzed data; and P.S., S.P., M.Y.E.-N., and G.J.J. wrote
the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Published under the
PNAS license
.
1
P.S. and S.P. contributed equally to this work.
2
To whom correspondence may be addresse
d. Email: mnaggar@usc.edu or jensen@
caltech.edu.
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1718810115/-/DCSupplemental
.
Published online March 19, 2018.
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the Mtr pathway, in which electrons are transferred from the
periplasmic decaheme cytochrome MtrA to the outer membrane
decaheme cytochrome MtrC through the transmembrane porin
MtrB (22, 23). Under conditions of direct cell surface contact
with minerals or electrodes, MtrC (and a partnering decaheme
cytochrome OmcA) can transfer electrons directly to these solid
EAs (24). The EET rate from the surface-exposed cytochromes
to such external surfaces can also be enhanced by interactions
with secreted flavins that function either as cytochrome-bound
cofactors (25
27) or soluble shuttles capable of interacting with
even more distant EA surfaces (28, 29).
Recent findings from live fluorescence light microscopy (fLM)
have hinted at the possible role of
Shewanella
OM extensions as
bacterial nanowires that transport respiratory electrons to EAs
micrometers away from the cell (30). First, the production of
OM extensions has been shown to correlate with an increase in
the cellular reductase activity (30). Second, the thickness of dried
OM extensions (two collapsed, 5-nm
thick lipid bilayers)
matches the thickness of dried and fixed conductive appendages
from
Shewanella oneidensis
(
10 nm) (21). Third, immunofluo-
rescence measurements have shown that the
S. oneidensis
mul-
tiheme cytochromes MtrC and OmcA localize along these OM
extensions (30). Importantly, the same multiheme cytochromes
have been shown to be essential for the solid-state conductance
of dried and fixed
S. oneidensis
appendages (21). Although these
multiple lines of evidence point to the ability of
S. oneidensis
OM
extensions to play a role in ET, direct conductance measure-
ments were demonstrated only on dry samples where the dis-
tribution and conformation of the ET components may not be
the same as in vivo (21). Additionally, outer membrane vesicles
(OMVs), structures similar to OM extensions, have been found
to be involved in various other functions including pathogenesis,
microbial interactions, and survival during stress conditions (31).
Therefore, to understand the extent to which
S. oneidensis
OM
extensions can carry electrons will require direct in vivo ET
measurements, challenging experiments due to the difficulty in
controlling growth and positioning of OM extensions to interface
electrodes. However, ultrastructural studies of the native con-
figuration of ET components, such as presented in this paper,
can provide useful information on the potential ET properties of
OM extensions.
So far, the diffraction-limited resolution of fLM has precluded
visualization of the macromolecular details of the OM extension
and its cytochrome distribution (30). Many other details remain
unclear, including formation and stabilization mechanisms, as
well as the processes underlying the large morphological varia-
tion and dynamic nature of these filaments. Furthermore, it has
been challenging to distinguish OM extensions from other fila-
ments (flagella, pili, and dehydrated extracellular polymeric
substances) (32, 33). Here, we use electron cryotomography
(ECT) to capture near-native images of OM extensions from
S. oneidensis
MR-1. ECT can deliver high-resolution 3D struc-
tural details of cellular structures. By capturing the specimen in a
thin layer of vitreous ice, structures of interest are preserved in a
fully hydrated and essentially native state (34).
We have developed a unique experimental setup allowing
bacteria to form OM extensions on an electron microscopy (EM)
grid inside a perfusion flow imaging platform. Using fluorescent
membrane staining, we monitored OM extension growth in real
time by fLM and subsequently located and imaged the same
structures by ECT. We discuss the challenges involved in
retaining the fragile OM extensions for EM imaging and the
methodology we developed to address these sample preparation
issues. Our fLM and ECT results reveal the vesicular nature of
S.
oneidensis
OM extensions and shed light on a potential mecha-
nism for their stabilization as OMV chains. The high resolution
of ECT reveals the positions of periplasmic and OM multiheme
cytochromes under near-native conditions. We discuss how these
structural measurements inform and help refine proposed
models (30, 35, 36) for long-distance ET.
Results
Conditions for Reliable OM Extension Production for ECT.
While
OMVs and OM extensions have previously been described in
both planktonic and surface-attached
Shewanella
cultures using
various methods such as EM, atomic force microscopy (AFM),
and fLM (21, 30, 37, 38), there has not been an extensive ex-
ploration of the optimal culturing and sample preparation
workflows most suitable for detection of these structures. Here
Inlet
Outlet
EM Grid
Glass
Coverslip
Perfusion Chamber
Vacuum Line
A
C
Microscope
Objective
Glass
Coverslip
EM Grid
O-rings
Shewanella
cell
Perfusion
Chamber
Inlet
Outlet
O-rings
Perfusion
Chamber
Body
Perfusion Chamber
Body
Inlet
Reservoir
(Medium)
Cells
Bubble
Trap
Perfusion
Chamber
Body
Outlet
Reservoir
Inlet
Outlet
Glass
Coverslip
Fluorescence
Microscope
Vacuum
Line
Inlet
Outlet
Perfusion Chamber
Body
Perfusion Chamber
EM Grid
Vacuum Line
Glass
Coverslip
O-rings
B
D
Fig. 1.
Schematic and actual images of the perfu-
sion flow imaging platform (objects not drawn to
scale). (
A and B
) Cross-sectional (
A
) and 3D (
B
) views
of the perfusion flow imaging platform. An electron
microscopy (EM) grid is glued to a glass coverslip that
seals the perfusion chamber.
S. oneidensis
cells in-
jected into the sealed chamber attach to the grid
surface and are sustained by a continuous flow of
the medium. Cells are labeled with the fluorescent
membrane dye FM 4-64FX and monitored in real
time for OM extension growth using an inverted
fluorescent microscope placed under the perfusion
chamber. (
C
and
D
) A 3D schematic (
C
) and image (
D
)
of the perfusion chamber interior with an attached
EM grid.
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we utilized negative stain transmission electron microscopy
(TEM) and ECT to assess both culturing and sample preparation
steps that lead to robust formation, preservation, and detection
of OM extensions. These steps are summarized in
Fig. S1
.
We first tested liquid cultures of
S. oneidensis
MR-1, either
from continuous-flow bioreactors (chemostats) operated under
O
2
-limited conditions (21, 30, 37) or from batch cultures (
SI
Materials and Methods
) by visually assaying for OM extension
formation by EM. Despite the presence of membrane blebs and
OMVs, longer OM extensions were rarely detected by either
negative stain TEM or ECT under our cultivation conditions,
even when fixed with glutaraldehyde to potentially stabilize the
structures (
Figs. S2
and
S3
). Separate imaging with scanning
electron microscopy (SEM) revealed an abundance of filaments,
but SEM
s lower level of structural detail makes it difficult to
distinguish the target OM extensions from other filaments such
as pili, flagella, and filamentous polymeric substances.
Because OM extensions in liquid cultures were only rarely
observed by both ECT and negative stain TEM, we next tested
surface-attached cultures. Building on our previous work utiliz-
ing coverslip-attached cultures to reveal the composition of
S. oneidensis
OM extensions (30), we developed a method for
monitoring their growth directly on EM grids inside a perfusion
flow imaging platform by fLM (Fig. 1). While extensions were
seen abundantly by fLM, very few structures remained intact
until the final step of either negative stain TEM or ECT workflow,
whether unfixed or fixed with formaldehyde (
Figs. S4
and
S5
).
This suggests that OM extensions are fragile structures that need
to be stabilized for TEM imaging. Fortunately, we found that
fixation with glutaraldehyde stabilized the extensions, enabling
us to reliably visualize the struc
tures by correlative light and
electron microscopy (CLEM) (
Fig. S6
and
Movies S1
and
S2
).
We conclude that (
i
) OM extensions are more frequent and
consistently present in surface
-attached cultures compared
with liquid cultures under our experimental conditions,
and (
ii
), although abundantly pr
oduced in surface-attached
samples, OM extensions are fra
gile structures that are easily
disrupted unless preserved by glutaraldehyde fixation for
TEM imaging.
Live Fluorescence Microscopy of OM Extension Growth on EM Grids.
Building on our previous work, we developed an optimized
perfusion flow imaging platform setup consisting of a microliter-
volume laminar perfusion flow chamber placed on an inverted
fluorescence microscope, with an EM grid-attached glass cov-
erslip sealing the chamber (Fig. 1).
S. oneidensis
cells are then
introduced into the chamber, where they attach to the surface of
the EM grid, and sterile media are flowed into the chamber
throughout the experiment. Using this setup, we observed
the formation of OM extensions live on the EM grid surface with
the fluorescent membrane dye FM 4-64FX. Cells were located
1
3
2
1
2
3
Brightfield
t=0 min
t=5 min
t=10 min
t=20 min
t=30 min
t=40 min
t=60 min
4
4
Fig. 2.
Live in vivo observation of the formation of
S. oneidensis
OM ex-
tensions (white arrows) on an EM grid. (Scale bar: 5
μ
m.)
Inset
is an overlap
of red fluorescence and reflective brightfield channels, revealing both the
holey carbon film coating the EM grid and the fluorescently labeled cells
attached to it.
Movie S3
is a time-lapse movie of this. (
1
,
2
, and
3
) Enlarged
views of boxed regions from the main panel. (Scale bars in
1
,
2
, and
3
:5
μ
m,
5
μ
m, and 2
μ
m, respectively.) (
4
) Time-lapse images of the growth of a single
OM extension from boxed region
4
in the main panel.
t
=
0 min is an arbi-
trary starting time point. (Scale bar: 5
μ
m.)
A
BCDE
Fig. 3.
Targeting dynamic OM extensions of
S.
oneidensis
for ECT using correlative light and elec-
tron microscopy. Target locations on fixed and
plunge-frozen electron microscopy grids, from the
perfusion flow imaging platform, were imaged by
ECT, revealing the OMV chain morphology of the
OM extensions. (
A
E
) Representative images from
ECT, with corresponding fLM image (
Insets
). (ECT
scale bars, 100 nm; fLM scale bars, 2
μ
m.) White
dotted boxes in the fLM images indicate the corre-
sponding approximate regions imaged in ECT. The
ECT images shown are tomographic slices from 3D
reconstructions (
Fig. S6
and
Movies S1
and
S2
).
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Subramanian et al.
relative to grid holes by fLM (Fig. 2 and
Movie S3
)toallowreg-
istration with subsequent EM imaging and thus enabling CLEM.
ECT Reveals OM Extensions Are Dynamic Chains of Interconnected
OMVs.
For ECT, grids from the perfusion flow imaging plat-
form were removed, plunge frozen, and transferred to the elec-
tron microscope, where the fLM-identified OM extensions were
located and imaged (Fig. 3). ECT images confirmed that ap-
pendages observed in fLM are in fact OM extensions, with the
two leaflets of the lipid bilayer clearly resolved along their length
(Fig. 4
A
and
B
). Cryotomograms revealed OM extensions to be
chains of interconnected OMVs in both unfixed (Fig. 4
C
) and
fixed samples (Fig. 4
D
G
). Previous fLM and AFM work
showed that OM extensions cover a range of morphologies from
apparently smooth tubes to clearly distinguishable OMV chains
(30). Here, with the higher resolution of ECT, we observed that,
with the exception of one smooth structure (
Fig. S7
), all OM
extensions including those t
hat appeared smooth in fLM were
distinguishable as OMV chains (Figs. 3 and 4). The images also
captured vesicle budding (Fig. 4
B
), a process that underlies the
initial stage of OMV production (39). Importantly, ECT allowed us
to clearly distinguish between pili, flagella, and OM extensions
the three known extracellular appendages in
S. oneidensis
(Fig. 4
D
G
and
Movies S4
and
S5
).
Electron-dense regions were observed at the junctions con-
necting neighboring vesicles throughout the length of the OM
extensions in both fixed and unfixed samples (Fig. 5
A
,
Fig. S8
,
and
Movie S6
). This finding points to yet unknown molecules
that potentially facilitate the constriction of the membrane to
allow OMV connections and is consistent with the fLM obser-
vations of OM extensions as dynamic structures capable of
growth, shrinking, and reversible transition between OMV chain
and individual vesicle morphologies (Fig. 5
B
and
C
and
Movies
S7
S10
). Fig. 5
D
provides a model to visualize how the junction
densities seen in ECT, when added or removed, may account for
the dynamic transitions in vesicle chains observed in fLM.
Distribution of Multiheme Cytochromes Along OM Extensions.
In
S. oneidensis
, previous immunofluorescence measurements have
shown that the OM cytochromes MtrC and OmcA localize along
the length of OM extensions (30). Additionally, the same cyto-
chromes were shown to be essential for solid-state conductance
of fixed and dried appendages consistent with OM extensions
(21, 30). The packing density of these cytochromes is crucial in
determining the potential mechanism of ET along OM exten-
sions, but has remained unknown. Here, using ECT, we observed
electron-dense particles on the interior and exterior of the OM
extensions. We confirmed that the observed particles correspond
to periplasmic and OM cytochromes by imaging OM extensions
from a mutant,
Δ
Mtr/
Δ
mtrB
/
Δ
mtrE
(40), lacking genes encoding
eight identified functional
S. oneidensis
periplasmic and OM
cytochromes. Our results showed a significantly higher interior
and exterior particle density in the wild type compared with the
mutant (Fig. 6 and
Movie S11
), confirming that a majority of the
densities in wild-type OM extensions are indeed cytochromes. In
addition, utilizing the OM extension with the highest number of
densities (Fig. 7
A
), we overlaid available structures of the deca-
heme cytochromes MtrA (41) and MtrC (26) on representative
interior and exterior densities, respectively, and found a similarity
in overall shape and size of these structures to the observed EM
densities (Fig. 7
B
).
C
-unfixed
AB
DFG
E
OME
OM
IM
IL
OL
OM
IM
BV
OME
F
P
F
OME
F
OME
OME
F
OME
OL
IL
IL
OL
PG
PG
Fig. 4.
ECT images of
S. oneidensis
OM extensions.
(
A
) OM extension membrane bilayer is clearly re-
solved.
Inset
is enlarged view of boxed region with
the inner and outer leaflets indicated with arrows.
(Scale bar, 100 nm;
Inset
scale bar, 10 nm.) (
B
)A
budding vesicle emerging as an extension of the
cellular outer membrane. A similar process perhaps
underlies the initial stages of OM extension forma-
tion. (Scale bar: 50 nm.) (
C
) OM extension from an
unfixed chemostat sample exhibits identically
branched OMV chain morphology as observed in
both unfixed and fixed samples from the perfusion
flow imaging platform. (Scale bar: 100 nm.) (
Figs. S5
and
S9
and
Movie S13
.) (
D
) An OM extension, a fla-
gellum, and a pilus next to each other, allowing di-
rect comparison of their sizes and morphologies,
indicating that ECT facilitates the identification and
distinguishability of different extracellular append-
ages in
S. oneidensis
. (Scale bar: 100 nm.) (
Movie S4
.)
(
E
G
) ECT reveals OM extensions are of varying thick-
nesses and degrees of tubulation. Next to each OM
extension is a flagellum that can act as a molecular
marker for comparison of varying OM extension di-
mensions. (Scale bar: 100 nm.) (
Movie S5
corresponds
to
F
.) BV, budding vesicle; F, flagellum; IL, inner leaflet;
IM, inner membrane; OL, outer leaflet; OME, OM ex-
tension; P, pilus; PG, peptidoglycan.
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We marked all of the observed interior and exterior densities
along the OM extension as model points and reconstructed 3D
models of both the OM extension and the cytochromes (Fig. 7
C
and
Movie S12
). The model allowed us to calculate the distance
of each cytochrome from its nearest neighbor and thus in-
vestigate the possible ET mechanism along OM extensions. The
observed density distribution fell in one of three categories:
patches where the densities were almost continuous and in-
distinguishable from one another (Fig. 7
D
and
E
), sections
where the exterior and interior densities clustered closely but
were distinguishable from one another (Fig. 7
F
and
G
), and
regions where the densities were farther apart (Fig. 7
H
). In
summary, we did not observe a c
ontinuous crystalline-like
packing of densities along the entire OM extension length. In-
stead, the OM and periplasmic densities were distributed over a
range of center-to-center spacings, from 4.9 nm to 32.5 nm and
from 5.0 nm to 29.0 nm, respectively (Fig. 7
I
). This distribution
of densities suggested an ET model that supplements direct
electron hopping between close cytochromes in tightly packed
sections with physical diffusion of cytochromes to bridge
larger gaps.
Calculations Suggest Maximum Overall ET Rate Is Achieved with a
Combination of Cytochrome Physical Diffusion and Direct Electron
Hopping.
To investigate the impact of cytochrome density on
ET along OM extensions, we used the Blauch
Saveant model
(42) that accounts for mobility of redox carriers in addition to
direct electron hopping between redox carriers in the membrane.
The relative contribution from redox carrier physical diffusion
and direct hopping to the overall ET rate is determined by the
ratio
t
e
/
t
p
(42), where
t
e
and
t
p
are the time constants for electron
hopping and physical motion of redox carriers, respectively. With
decaheme OM cytochromes as the redox carriers in OM exten-
sions, and using 3
μ
m
2
/s as a representative value for the physical
diffusion coefficient of integral membrane proteins of similar size
(
D
phys
)(43),
t
p
is estimated to be
3
×
10
6
s(
SI Materials and
Methods
). In addition, using the electron residence time in the
heme chains of the individual cytochromes, estimated from cal-
culated and measured electron flux through MtrF (10
4
s
1
)(44,
45) and MtrCAB (24),
t
e
can be estimated to be
10
4
s and hence
t
e
/
t
p
to be
30. This relatively high value of
t
e
/
t
p
(i.e.,
t
e
/
t
p
>>
1)
justifies a mean-field approach developed by Blauch and Saveant
(42), leading to a simple expression for the apparent diffusion
coefficient (
D
ap
) (42),
D
ap
=
D
phys
ð
1
X
Þ
f
c
+
D
e
X
,
[1]
where
D
phys
is the redox carrier physical diffusion coefficient,
D
e
is the electron hopping diffusion coefficient which can be calcu-
lated using
t
e
(
SI Materials and Methods
),
f
c
is the correlation
factor, and
X
is the fractional loading of redox carriers in the
membrane which can be calculated using particle densities
extracted from the cryotomograms (
SI Materials and Methods
).
Therefore, for OM extensions,
D
ap
is estimated to be
3
×
10
8
cm
2
/s. In addition, the electron flux through an OM extension (
J
)
can be calculated by (42)
J
=
D
ap
C
x
,
[2]
where
C
is the concentration of the reduced redox carriers and
x
is the position along the length of the OM extension. The result-
ing overall ET rate for an idealized 1-
μ
m
long, 100-nm
diameter
OM extension is shown in Fig. 8, where MtrC molecules are
assumed to be the electron carriers (
SI Materials and Methods
).
Discussion
Here we show high-resolution images of OM extensions in
S.
oneidensis
, using ECT. We found the OM extensions to be OMV
chains possibly stabilized by constriction densities at the junc-
tions. Bacterial membrane extensions have been reported in
multiple organisms:
nanopods
in
Comamonadaceae
including
Delftia
(46),
outer membrane tubes
in
Francisella novicida
(47),
periplasmic tubules
in
Chlorochromatium aggregatum
(48),
B
C
A
- Fixed
D
Junction density
MtrC
MtrA
OM
IM
t=0 min
20 min
t=0 min
25 min
45 min
110 min
140 min
145 min
160 min
75 min
110 min
120 min
130 min
140 min
Fig. 5.
Proposed model for the formation and sta-
bilization of OMV chains. (
A
) ECT image of a chem-
ically fixed OM extension reveals the presence of
densities at junctions that connect one vesicle to the
next along the OMV chain (white dashed boxes).
While all of the junction densities are not visible in
the tomographic slice in
A
,
Movie S6
is a 3D re-
construction of the same OM extension revealing the
densities present at every junction. In addition,
densities possibly related to decaheme cytochromes
can be observed on the interior and exterior of the
OM along the extension (arrows). (Scale bar:
100 nm.) (
Fig. S8
.) (
B
and
C
) Time-lapse fluorescence
images recorded in real time in the perfusion flow
imaging platform monitoring the growth and
transformation of an OM extension from an appar-
ently long filament (OMV chain morphology) to a
single large vesicle (
B
, indicated by arrows) in
S. oneidensis
Δ
flg
(a mutant strain lacking flagellin
genes). (
Movie S7
.)
Movie S8
shows OM extensions
from wild-type cells also exhibiting a similar behav-
ior to
Δ
flg
and a large vesicular morphology to
an apparently smoother filament (OMV chain
morphology) (
C
, indicated by arrows) in wild-type
S. oneidensis
MR-1 cells. (
Movies S9
and
S10
). The
cells and the OM extensions in
B
and
C
are stained by
the membrane stain FM 4-64FX. (Scale bars in
B
and
C
:5
μ
m and 2
μ
m, respectively.) (
D
) Schematic
depicting a hypothesis for the formation and stabi-
lization mechanism of OMV chains: Junction densi-
ties on the interior of the OM extension facilitate the constriction of the membrane, enabling the formation of an OMV chain. These constriction densi
ties can
be removed or added to facilitate transformation of an OMV chain to a large vesicle or vice versa as observed in
B
and
C
, respectively.
E3250
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www.pnas.org/cgi/doi/10.1073/pnas.1718810115
Subramanian et al.
membrane tubules
in
Salmonella typhimurium
(49),
nano-
tubes
in
Bacillus subtilis
(50) and connecting
Escherichia coli
cells to each other and to
Acinetobacter baylyi
cells (51), and
connecting structures
that allow exchange of material between
Clostridium acetobutylicum
and
Desulfovibrio vulgaris
cells (52).
However, membrane extensions in the form of OMV chains,
similar to those reported here, have only recently been discov-
ered and much remains unknown about their formation mech-
anism and specific function (53). In the gram-negative
Shewanella vesiculosa
(54) and
Myxococcus xanthus
(55, 56) and
the gram-positive
B. subtilis
(57), membrane extensions in the
form of OMV chains have been observed using cryo-EM with
implications for cell
cell connections in the latter two exam-
ples. While the
S. oneidensis
OM extensions are proposed to
function as electron conduits (30), their structural similarity to
these previous reports highlights the significance of imaging
these structures as a model system to study the formation of
OMV chains.
To find a condition that consistently and frequently produced
intact OM extensions for ECT imaging, we systematically tested
different methods of growth and sample preparation conditions,
as summarized in
Fig. S1
. We found that our optimized perfusion
setup (Fig. 1) was best suited for the formation (Fig. 2), sub-
sequent CLEM (Fig. 3), and high-resolution cryotomography of
OM extensions (Fig. 4). The OMV chain morphology exhibited
by these OM extensions is unlikely to be an artifact of fixation
since we also observed a similar OMV chain architecture in OM
extensions from unfixed samples (Fig. 7
A
and
Fig. S5
). While
flagella and pili were identified as smooth filaments measuring
10 nm and
3 nm in thickness, respectively, OM extensions
varied in thickness typically from
20 nm to 200 nm (Fig. 4
D
G
), depending on the size and extent of tubulation of the con-
stituent OMVs. Typically, there was an inverse relationship
between OM extension length and its constituent OMV size. The
measured thickness of hydrated OM extensions in ECT is dif-
ferent from the previously reported AFM measurements of
10 nm for air-dried conductive appendages (21, 58, 59). This is
consistent with the finding that the appendages are OM exten-
sions (30) because, in AFM, dehydration causes OM extensions
to collapse to an
10-nm thickness, roughly corresponding to
two lipid bilayers, while ECT preserves samples in a frozen-
hydrated state, leading to more accurate estimates of native
thickness. In addition to changing the OM extension thickness,
dehydration will alter the cytochrome conformation and packing
along OM extensions, which could significantly impact their
electron-carrying capabilities. An interesting feature we observed
is the ability of the vesicle chains to branch (Fig. 7
A
,
Fig. S9
, and
Movie S13
), which may offer the advantage of increasing the
Surface Density
(particles/μm^2)
Exterior and Interior
Particles P=2.8e-4
Exterior Particles
P=4.8e-5
Interior Particles
P=7.5e-3
GH I
Fig. 6.
Difference in surface density along OM ex-
tensions between
S. oneidensis
wild type and a mu-
tant lacking all identified functional OM and
periplasmic cytochromes (
Δ
Mtr/
Δ
mtrB
/
Δ
mtrE
) (40).
(
A
and
B
) Representative ECT images of OM exten-
sions from wild-type and mutant strains, respectively,
revealing electron-dense particles on the interior and
exterior of the membrane. White dashed line indi-
cates two different slices have been combined to
provide the best possible view of OM extension.
(Scale bar: 100 nm.) (
C
and
D
) Enlarged views of the
vesicle from the boxed regions in
A
and
B
,re-
spectively, with membrane (cyan line), interior par-
ticles (green circles), and exterior particles (red
circles) labeled as model points. (Scale bar: 20 nm.) (
E
and
F
) The 3D reconstructions of the vesicles in
C
and
D
, respectively. Meshed view of the membrane is
generated and all of the observed interior and ex-
terior densities are shown as model points in 3D.
(Scale bar: 20 nm.) (
Movie S11
.) (
G
I
) Surface density
(in particles/
μ
m
2
) of total, exterior, and interior par-
ticles in the wild-type (
n
=
8) and cytochrome mutant
(
n
=
5) OM extensions (one vesicle analyzed per OM
extension). Statistical significance is determined by
P
values from unpaired one-tailed Student
s
t
tests.
Error bars represent one SD around the mean.
Subramanian et al.
PNAS
|
vol. 115
|
no. 14
|
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MICROBIOLOGY
BIOPHYSICS AND
COMPUTATIONAL BIOLOGY
PNAS PLUS